This invention relates to methods for DNA genotyping and gene mapping whereby nucleotide variations, mutations and polymorphisms are quickly and accurately detected by the enzymatic methylation of DNA using sequence-specific DNA methyltransferases. In particular, a novel method is. described in which DNA can be xe2x80x9cpainted xe2x80x9d at specific sites using radiometric or immunochemical detection. This novel methodology has been identified by the term xe2x80x9cDNA Paintxe2x80x9d. DNA methyltransferases can be used alone, or in combination with restriction endonucleases, to (a) diagnose diseases of plants, animals, or humans; (b) map genes; or (c) detect genetic mutations or polymorphisms.
Starting in the early 1970""s with the emergence of the recombinant DNA methods and rapid DNA sequence technologies that followed soon thereafter, scientists have relied upon restriction endonucleases to study the genetic makeup of plants and animals. The medical field has shown great advancements through the use of this molecular genetic revolution through the identification of nucleic acid variations, mutations, and polymorphisms within genes and over entire genomes. In addition, the ability to identify and recombine specific alleles for genes of interest allow researchers to create improved livestock or plant varieties much more quickly than traditional breeding methods have allowed.
The discovery of sequence-specific DNA cutting enzymes, restriction endonucleases, made the development of recombinant DNA technologies possible. Over 2,000 different sequence-specific endonucleases have been characterized, of which about 200 are available. R. J. Roberts and D. Macelis, xe2x80x9cREBASExe2x80x94Restriction enzymes and methylasesxe2x80x9d, Nusleic Acids Res., 24:223-235 (1996), incorporated herein by reference. These restriction endonucleases have been widely used in RFLP mapping, DNA fingerprinting, gene mapping, to detect mutations responsible for heritable human diseases and polymorphisms associated with traits of interest in animal and plant breeding programs, and to diagnose infectious disease agents or viral, bacterial, or fungal origin.
Gene mapping, DNA fingerprinting, and RFLP technologies routinely exploit the variations and mutations between the genomes of different individuals or species or varietal populations. When a point mutation, insertion, or deletion, alters the DNA sequence within a genome, the restriction endonuclease enzyme can detect these changes in nucleic acid sequence if the change creates or destroys a restriction endonuclease recognition sequence. Restriction endonucleases are sequence-specific DNA-cutting enzymes which recognize a specific nucleic acid sequence pattern and cleave DNA strands at specific locations. The restriction enzymes most often used in gene mapping and DNA fingerprinting technologies are Type II bacterial restriction enzymes, most of which recognize 4 to 6 base pair recognition sequences. Therefore, on average, most DNA restriction fragments are 44 to 46 (256 to 4096) base pairs (bp) long. When a mutation or variation occurs within one of these DNA sequence recognition sites, those restriction enzymes which cleave at that recognition sequence will no longer be able to cut at that site if the site is destroyed. Conversely, if a DNA sequence recognition site is created, the restriction enzyme for that site will be able to cleave the DNA where it could not previously. It is this variation in the presence and absence of restriction enzyme recognition sites within the genomes of different individuals that allows for DNA mapping and genetic fingerprinting using restriction enzymes. (Botstein et al., xe2x80x9cConstruction of a genetic linkage map in man using restriction fragment length polymorphismsxe2x80x9d, Am. J. Human Genetics, 32:314-331 (1980); R. White and J. J. Lalouel, xe2x80x9cChromosome Mapping with DNA Markersxe2x80x9d, Scientific American, 258:40-48 (1988).
While the advent of the restriction enzymes has revolutionized molecular genetics, it has not come without its inherent weaknesses. In order to visualize the changes in restriction endonuclease target sites (and to therefore define a restriction isotype or an allelic fingerprint), the DNA fragments that result from restriction endonuclease cleavage must be separated by size. In short, if DNA is cut into pieces then the resulting pieces must be separated. Differences in fragment size create different morphological patterns, hence polymorphisms, for each individual or trait of interest. These polymorphic patterns (DNA fingerprints) are best observed when run out by electrophoresis on agarose or polyacrylamide gels.
Another limitation using bacterial restriction enzymes in RFLP mapping or genotyping is that most mutations or polymorphic sites in DNA do not occur within these relatively rare (4 to 6 bp) endonuclease recognition sequences. The present invention overcomes this limitation through the use of sequence-specific DNA Methyltransferases (MTases) which can recognize short 2 to 4 bp sequence recognition sites as well as longer 4 to 8 bp sequence recognition sites. In particular, 75% of point mutations responsible for all known heritable human diseases occur at CG sites. D. Cooper and H. Youssoufin, xe2x80x9cThe CpG dinucleotide and human genetic diseasexe2x80x9d, Human Genetics 78:151-165 (1988), incorporated herein by reference. Several DNA MTases recognize 2 bp CG sites, such as M.SspMQI, M.SssI, and M.DmtI of mouse (Okano, M., et al, xe2x80x9cCloning and Characterization of a Family of Novel Mammalian DNA (Cytosine-5) Methyltransferasesxe2x80x9d, Nature Genetics, Vol. 19, Jul. 1998, 219-220), human, Arabidopsis, and algal virus DNA MTases (Nelson et al., xe2x80x9cDNA Methyltransferases and Site-specific Endonucleases Encoded by Chlorella Virusesxe2x80x9d, in: G. P. Jost and H. P. Salusz HP, eds., DNA Methylation, Birkauser and Basel, pp. 186-211 (1993)). In contrast, no known restriction endonucleases recognize CG or any other dinucleotide sequences.
The present invention uses DNA MTases rather than restriction enzymes to overcome many of the above-mentioned problems. Each restriction endonuclease has a companion sequence-specific DNA methyltransferase (MTase) which has the same DNA recognition site. W. Arber and S. Linn, xe2x80x9cDNA Modification and Restrictionxe2x80x9d, Ann. Rev. Biochem. 38:467-500 (1969), incorporated herein by reference. Several hundred of these DNA MTase specificities are known (McClelland et al., xe2x80x9cEffect of site-specific modification on restriction endonucleases and DNA MTasesxe2x80x9d, Nucleic Acids Res. 22:3640-3659 (1994), incorporated herein by reference) and more are being discovered each year. In addition, as mentioned above, there are several DNA MTases which recognize 2 to 4 bp DNA sequences, such as the CG dinucleotide sequence, whereas there are no known restriction endonucleases with such short recognition sequences. As described in this patent application for the first time, it is possible to use methyltransferases rather than restriction enzymes to detect genetic polymorphisms and mutations at 2 to 8 bp sites within genomic DNA.
The present invention can utilize PCR amplification and sequence-specific DNA methylation to detect the presence or absence of specific DNA methyltransferase recognition sites.
DNA methyltransferases (MTases) catalyze the transfer of methyl groups from S-Adenosylmethionine (SAM) to specific sites in double-stranded DNA, yielding methylated DNA and S-Adenosylhomocysteine (SAH). 
If radioactive 3H-methyl-SAM is used as a substrate, then the number of methyl groups incorporated into DNA can be measured by trichloroacetic acid (TCA) precipitation of 3H-methyl-DNA, followed by liquid scintillation counting. 
This reaction is usually termed a xe2x80x9cSAM-dependent DNA methyltransferase reactionxe2x80x9d. However, it might be better termed a xe2x80x9cDNA-dependent SAM methyl transfer reactionxe2x80x9d, since the 3H-methyl groups are incorporated into DNA only if a sequence-specific MTase recognition site is present. If one or more DNA MTase sequence recognition sites are present, then the number of such sites can be measured quantitatively based on a linear increase in tritium counts per minute (cpm) of radiolabeled 3H-methyl DNA.
Sequence-specific DNA MTase enzymes have been used only sparingly in megabase mapping experiments. M. Nelson and M. McClelland, xe2x80x9cThe use of DNA MTase/Endonuclease enzyme combinations for megabase mapping of chromosomes by pulsed field gel electrophoresisxe2x80x9d, Methods in Enzymolog 216:279-303 (1992), incorporated herein by reference. These megabase mapping experiments utilize sequential multi-step DNA methylation and restriction enzyme cleavage reactions to create chromosome fragments in the size range from 50 to 2000 kilobases (kb) that are subsequently analyzed using pulse field gel electrophoresis (PFGE). See D. C. Schwartz and C. R. Cantor, 37 Cell 67 (1984); Gardiner et al., 12 Somatic Cell. Mol. Genet. 185 (1986); Cantor et al., 17 Ann. Rev. Biophys. Chem. 287 (1988). These experiments describe how MTases can be used to increase the apparent specificity of restriction enzymes. However, they do not describe how MTases can be used to xe2x80x9cpaintxe2x80x9d DNA at specific 2 to 8 bp sites using either radioactive (3H-methyl-DNA) or immunochemical detection. Furthermore, the techniques of the above cited references are normally carried out in situ on unsheared chromosomes embedded in agarose plugs and it has proven necessary to require: (1) that the purity of the MTases and the restriction endonucleases be critically controlled, (2) that the number of steps be kept at a minimum, and (3) that the reaction conditions be defined which are compatible with PFGE separation. This technique includes the inherent drawbacks of PFGE such as the time and effort required to prepare high molecular weight substrates and to run pulsed-field gels. The present invention overcomes these problems by creating smaller fragments of genomic DNA or utilizing PCR-type techniques, thus eliminating the need for agarose embedded in situ reactions or completely eliminating the need for electrophoresis. Further, the present invention allows for much finer mappingxe2x80x94down to the nucleotide levelxe2x80x94and exploitation of this information for such uses as diagnostic testing in clinical settings.
In general, DNA MTases have been used neither to map genes nor to detect mutant alleles. The present invention shows how DNA MTases can be used to radio- or immuno-chemically xe2x80x9cpaintxe2x80x9d DNA in order to identify polymorphic sites or mutations. Using DNA MTases, one can determine genotypes with significant improvements in speed, accuracy and specificity over RFLP mapping and other methods utilizing restriction endonucleases. In short, the methods of the present DNA MTase invention are extremely fast, do not require gel electrophoresis or nucleic acid hybridization, and result in simple quantitative and/or qualitative assays. As a result, DNA MTase genotyping of the present invention lends itself to automation. Rapid automated PCR-based MTase genotyping may be especially useful in high throughput diagnostic laboratories or in situations where fast, easy, reproducible results are required, such as clinical laboratory or hospital settings.
The present invention does not require that DNA fragments be separated on agarose gels. Southern transfers, nucleic acid hybridization or radioactive labeling are not required, although these are possible uses of DNA MTases in certain embodiments of the present invention. Most importantly, since MTases do not cut the sugar-phosphate backbone of DNA, there are no DNA fragments that need to be separated. The present invention overcomes the lengthy and laborious dependence on gels and Southern transfers. The present invention allows for quick and quantitative analysis without the need to separate by fragment size using gels or time-consuming Southern transfer techniques. Certain embodiments of the present invention do not require the use of restriction enzymes at all. This independence from endonucleases allows the diagnostician or researcher to avoid erroneous results due to incomplete restriction enzyme digestions. The present invention also allows for the use of very short PCR products (30-100 bp). Since the PCR reaction cycle times are shortened to a few seconds, the concentrations of amplified DNA are increased, resulting in improved signal-to-noise. Thus the present inventive methods utilizing DNA MTases for genotyping are faster, more accurate, and easier to perform than the methods known to those skilled in the art utilizing bacterial restriction enzymes.
It is an object of the present invention to provide a method of genotyping DNA utilizing a DNA methyltransferase in conjunction with a PCR amplicon so that a quick and accurate determination of a mutation or nucleic acid variation can be determined.
A second object of the invention is to provide a method utilizing a DNA methyltransferase to genotype DNA whereby the method of detection relies on immunochemical (non-radioactive) detection, so that radioactive label is not required.
A third object of the invention is to provide a method of genotyping DNA utilizing a DNA methyltransferase without the need for agarose gel fractionation of DNA or Southern transfer.
A fourth object of the invention is to provide a method of ordered genetic maps of PCR-amplified DNA utilizing biotinylated primers and combinations of DNA methyltransferases and endonucleases. In other words, not only can the presence of MTase sites be determined, but their positions relative to the 5xe2x80x2 biotinylated end can be located.
A fifth object of the invention is to provide a method of DNA genotyping utilizing DNA methyltransferases whereby the determination of mutation or variation can be accomplished in a single-container reaction with no need for removal of reaction precursors, thus allowing for an even faster turnaround time.
A sixth objective of the invention is to provide a method utilizing DNA methyltransferases to identify individual alleles associated with disease traits or traits of economic importance and which could further be automated for fast, accurate and economic results.
A seventh objective is to provide a genotyping method which relies upon detecting the presence or absence of DNA MTase recognition sites, so that detection of specific DNA regions or alleles can be automated.
Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objects and advantages of the invention will be obtained by means of the instrumentalities and combinations, particularly pointed out in the appended claims.
To achieve the objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the present invention provides a method for exploiting DNA MTases, ability to methylate DNA at specific nucleic acid sequences. The sequence-specific DNA MTases can be used to detect the presence of specific recognition sites in nucleic acid molecules. Known or unknown sequences can be genotyped using DNA MTases. For example, MTases can be used to detect genetic mutations or polymorphisms or simply to gain DNA sequence information of unknown genes. The ability, or inability, of these DNA MTases to methylate at the sites of mutation or polymorphism are then detected by means which can include immunochemical or radiometric detection methods. Upon detection of the sites of methylation, DNA sequence information can be compiled, analyzed and referenced.
Numerous immunochemical detection methods are known to those of ordinary skill in the art and are readily available through commercial sources such as Fisher Scientific, Pittsburgh, Pa.; Boehringher Mannheim Corp., Indianapolis, Ind.; and Vector Laboratories, Burlingame, Calif. These immunodetection methods rely upon haptens such as biotin, digoxigenin, or those detected by antibodies or lectins conjugated with enzymes such as alkaline phosphatase, horseradish peroxidase, or xcex2-Galactosidase. See, Christopher Kessler, xe2x80x9cThe digoxigenin:anti-digoxigenin (DIG) technologyxe2x80x94a survey on the concept and realization of a novel bioanlytial indicator systemxe2x80x9d, Molecular and Cellular Probes, 5:161-205 (1991). Immunochemical detection methods can include, among others, labeling systems which utilize chemiluminescence substrates, chromogenic substrates, rhodamine, and fluorochrome-labels such as fluorescein isothiocyanate and tetramethylrhodamine. Numerous radiometric detection methods are also known to those of ordinary skill in the art and readily available through commercial sources such as Amersham Lifesciences, Arlington Heights, Ill. and New England Nuclear, Boston, Mass. However, the use of sequence-specific DNA MTases in vitro, followed by immunochemical detection of methylated DNA has not been previously described. Furthermore, none of these earlier methods allow DNA to be internally labeled at defined 2 to 8 bp sites.
The methods of this invention include as preferred embodiments in which these DNA MTases are utilized: (1). a radiometric 3H-methyl incorporation assay, theoretically also 3H3C11-, position-labeled methyl assay; (2) a non-radioactive immunochemical detection; (3) a combination of sequence-specific DNA methylation, restriction enzyme digestion, and Southern blotting (xe2x80x9cDNA Paintxe2x80x9d); and (4) an anchored PCR format in which biotinylated primers are used in combination with DNA MTases and endonucleases. Importantly, this last format allows ordered maps to be rapidly constructed without the need for either electrophoresis or nucleic acid hybridization.
In one embodiment, the invention comprises a method for genotyping an unmethylated DNA fragment produced by PCR amplification. Using PCR primers that flank the region of interest, enough DNA is amplified to conduct a series of sequence-specific DNA MTase and/or restriction endonuclease reactions. The design of PCR primers is known to those of skill in the art and are described in Mullis et al., The Polymeragg Chain Reaction, Burkhxc3xa4user Publishers, Boston, Mass. (1994), the disclosure of which is incorporated by reference. Amplified DNA is treated with selected DNA methyltransferases and radioactively-labeled-methyl-SAM. The radioactively labeled methyl group will thus be incorporated into a specific 2 to 8 bp site in the DNA. If appropriate, sufficient DNA may be put into reaction tubes to allow for digestion by selected restriction enzymes. The amount of radioactively labeled methyl DNA will be measured using radiometric assay methods well known to those of ordinary skill in the art. This technique will identify a DNA MTase genotype for each recognition sequence site within the PCR generated DNA fragment.
In a second embodiment of the invention, the genotyping of genes, or alleles of genes, associated with a disease condition, or a trait of economic interest in livestock or plants, can be determined utilizing DNA methyltransferases. DNA is first amplified using PCR primers that flank the gene, or segment of the genome, of interest. The amplified DNA is methylated with a radioactively labeled methyl group at a specific site known to vary from one allele to another. A sequence-specific DNA MTase can then be used to detect a defined genetic variation or mutation. Methylation of the DNA may be accomplished either at the same time as the amplification of the PCR fragment using thermostable MTases or following the amplification of the PCR fragment of interest if no thermostable methyltransferase is available for the site of interest. The presence or absence of the variation or mutation in question is determined by radiometric assay. If adequate signal-to-noise is achieved, then the wild type (a/a) state, heterozygous (a/b) state, or homozygous (b/b) state can be detected for that site.
In a third embodiment, the presence or absence (and the quantification of multiple sites) can be determined utilizing immunochemical detection rather than radioactive detection of methylated DNA. Detection of methylated DNA can be accomplished utilizing the above methods or other methods, as will be evident from this disclosure, by enzymatically incorporating non-radioactive methyl groups utilizing DNA methyltransferase. The resulting DNA can be spotted on to a fixed surface such as nitrocellulose or nylon membranes and adhered through routine methods known in the art. The DNA can also be separated by size on an agarose gel and transferred onto membranes using the Southern transfer technique. The membranes can then blocked to prevent background signal due to non-specific noise. In one example of this embodiment, a nylon membrane containing methylated DNA fragments are UV-irradiated and then incubated in the presence of anti-6xe2x88x92smethyladenine rabbit immune serum in buffered nonfat milk blocking solution. The filters are washed and treated with a solution containing alkaline-phosphatase conjugated goat anti-rabbit IgG or antiserum. After washing, NBT/NCIP substrate for alkaline-phosphatase is added. Methylated 6mA DNA is detected as blue spots or bands on a white nylon filter background.
In a fourth embodiment (sequence-specific xe2x80x9cDNA Paintxe2x80x9d Format), a combination of sequence-specific DNA methylation, restriction digestion, and Southern blotting technique allows methylated DNA fragments to be selectively stained. Such xe2x80x9cmethylase paintingxe2x80x9d of DNA fragments can be accomplished by digesting DNA with restriction endonucleases, and methylating the resultant DNA fragments with DNA methylases that have a different sequence specificity site. In general, DNA methylation is conducted prior to endonuclease digestion. However, the order can usually be reversed. The resultant fragments are then separated by size utilizing an agarose gel. After electrophoresis, the DNA is transferred from the gel to a membrane (nylon or nitrocellulose) using Southern transfer technique or other transfer methods known to those of ordinary skill in the art. The membrane-bound methylated fragment patterns are detected using radiometric techniques (if radioactively-labeled-methyl groups were incorporated) or immunochemical detection methods. This embodiment results in a DNA membrane or image exhibiting a unique pattern, or xe2x80x9cDNA Paintingxe2x80x9d, for each genome or DNA fragment of interest. Simply put in binary code, any particular DNA fragment is either radioactively or immunochemically labeled (1) or else it is not labeled (0). The presence or absence of methylated 2 to 8 bp DNA MTase recognition sites appear as colored bands or spots.
In a fifth embodiment, the invention comprises a method whereby the PCR fragment is anchored to a solid matrix, for example, a magnetic bead or Streptavidin SPA bead, so that radioactively labeled methyl groups can be counted using appropriate radiometric assays.